1. Field of the Invention
The present invention relates to the field of surrogate markers for the detection of stress in an organism. More specifically, the present invention relates to methods of use of 8-nitroguanine to detect stress in an organism, particularly as an early predictor of organ rejection in a transplant recipient. The present invention further relates to novel methods of synthesis of 8-nitroguanine and compositions comprising 8-nitroguanine.
2. Description of Related Art
Peroxynitrite is formed in the body by macrophages as part of the inflammation process (Yermilov et al., 1995a; Byun et al., 1999). Peroxynitrite can react with DNA to form 8-nitroguanine (Yermilov et al., 1995a, 1995b, 1996). It has been speculated that peroxynitrite may cause DNA or tissue damage, contributing to the multistage carcinogenesis process (Yermilov et al., 1995a; Douki et al., 1996; Spencer et al., 1996). Formation of 8-nitroguanine as a result of exposure to peroxynitrite could result in spontaneous depurination and single-stranded DNA breaks (Yermilov et al., 1995a), resulting in the release of 8-nitroguanine. Thus, 8-nitroguanine could potentially serve as a surrogate marker for stress in general and for inflammation-related stress in particular. More recently, it has been suggested that 8-nitroguanine does not spontaneously depurinate and that prior reports of spontaneous depurination were an artifact induced by non-physiological exposure to peroxynitrite (Tuo et al., 2000). The potential use of 8-nitroguanine as a surrogate marker for inflammation and stress, prior to the present invention, was thus uncertain.
Methods for non-invasive monitoring of stress, through the detection and quantification of 8-nitroguanine in body fluids such as blood, urine and sputum would be highly desirable. Such methods could provide, for example, a non-invasive method for predicting the likelihood of organ rejection in transplant recipients, or for detecting exposure to environmental stressors in the form of ionizing radiation, toxic chemicals or infectious agents like viruses and bacteria.
The development of such monitoring procedures would be facilitated by the availability of a low-cost method for production of 8-nitroguanine, which would be of use as a standard for calibration of monitoring systems. Present methods for the production of 8-nitroguanine are expensive and require the use of starting materials, such as 8-bromoguanine or peroxynitrite (Yermilov et al., 1995b; Spencer et al., 1996), that are highly toxic or that may not be readily available.
The present invention satisfies a long-standing need in the field, by providing a novel and low-cost method for production of 8-nitroguanine. In another embodiment, the present invention concerns methods for detecting and quantifying 8-nitroguanine in fluids, such as blood, urine or sputum. Further embodiments of the present invention concern compositions comprising 8-nitroguanine, made by the disclosed methods. Such compositions are of use as standards for calibrating equipment to detect 8-nitroguanine in samples.
In certain embodiments, the compositions and methods of the present invention are used for the detection and/or quantitation of exposure to environmental stressors such as ionizing radiation, toxic chemicals or infectious agents. In a preferred embodiment, the compositions and methods are of use for predicting the likelihood of organ rejection in transplant recipients. Such a non-invasive method for predicting the likelihood of organ rejection is superior to present methods that require biopsy samples from the transplanted organ.